Various biological preparatory techniques require immobilization of sample materials, such as cells, tissue, proteins, or nucleic acids, to a substrate prior to subsequent processing. Many of these biological materials of interest are anionic in nature, exhibiting net negative charge sites. One method of immobilizing these materials is to coat the target substrate with a chemical solution containing active ingredients that are cationic in nature, exhibiting a net positive charge. As the biological materials of interest to be immobilized exhibit net negative charge sites, the biological materials bind to the surface of the substrate through interaction with the net positive charge sites of the coating solution. This adhesion property of the coating solution allows the immobilization of sample material for subsequent processing.
The immobilization effect described above can be created through the use of coatings containing various active ingredients currently known in the art. For example, it is currently known to use coating agents, such as poly-1-lysine, 3-aminopropyl triethoxysilane, chrome alum gelatin, and egg white albumin. One of the most widely used of these known immobilization agents is poly-1-lysine (PLL).
PLL is a large polycationic homopolymer that exhibits a strong positive charge produced by the terminal amino groups of the lysine residue side chains all along the polymer. L-Lysine [(S)-2,6-diaminohexanoic acid] is an amino acid of the chemical structure shown below in formula (1).
The polymer PLL is a chain of 1-lysine monomer units attached through peptide bonds. The chemical structure for PLL is provided below in formula (2), wherein n is an integer representing the number of monomer units in the polymer chain.

While PLL is widely used as a polycationic polymer coating, substrates coated with PLL tend to lose their immobilization effectiveness over a relatively short time period. This decline in effectiveness over time is generally thought to be due to oxidation of the PLL side chain amine groups. The oxidized groups do not exhibit the net positive charge required for proper adhesion to the biological materials to be immobilized.
The effectiveness of PLL as an immobilization agent is also limited by its inherent chemical structure shown above in formula (2). As previously noted, the amino acid residues of the polymer are connected by peptide bonds (—CO—NH— bonds). These peptide bonds are highly vulnerable to cleavage by proteolytic enzymes, such as trypsin, and to general hydrolytic cleavage, such as through attack from a nucleophilic substance. Cleavage of the peptide bonds results in PLL molecules of substantially shorter chain length, as measured by the average molecular weight of the polymer. As the molecular weight of the PLL molecule is reduced through proteolytic cleavage, the immobilization capability of the molecule (i.e., its adhesive property) becomes greatly reduced.
Known immobilization agents, such as PLL, exhibit limited usefulness as a result of the chemical instabilities described above. Accordingly, substrates coated with the known agents also exhibit limited usefulness, particularly for long-term use or use after significant storage time. Given the limited stability of substrates coated with the known immobilization agents, it would be highly useful to have a pre-coated substrate that is coated with an immobilization agent that exhibits increased stability, particularly being useful for immobilizing a biological sample for observation.